1. Field of Invention
This invention relates to molecules useful as agonists of the insulin-like growth factors (IGFs), as well as IGF-like insulin molecules. More particularly, these molecules inhibit the interaction of an IGF or insulin with one or more of the IGF binding proteins. Such molecules can be used, for example, in any methods where the IGFs or insulins are used, for example, in treating hyperglycemic, obesity-related, neurological, cardiac, renal, immunologic, and anabolic disorders.
2. Description of Background and Related Art
The insulin-like growth factors I and II (IGF-I and IGF-II, respectively) mediate multiple effects in vivo, including cell proliferation, cell differentiation, inhibition of cell death, and insulin-like activity (reviewed in Clark and Robinson, Cytokine Growth Factor Rev., 7: 65-80 (1996); Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995)). Most of these mitogenic and metabolic responses are initiated by activation of the IGF-I receptor, an xcex12xcex22-heterotetramer closely related to the insulin receptor (McInnes and Sykes, Biopoly., 43: 339-366 (1997); Ullrich et al., EMBO J., 5: 2503-2512 (1986)). Both proteins are members of the tyrosine kinase receptor superfamily and share common intracellular signaling cascades (Jones and Clemmons, supra). IGF-insulin hybrid receptors have been isolated, but their function is unknown. The IGF-I and insulin receptors bind their specific ligands with nanomolar affinity. IGF-I and insulin can cross-react with their respective non-cognate receptors, albeit at a 100-1000-fold lower affinity (Jones and Clemmons, supra). The crystal structure describing part of the extracellular portion of the IGF-I receptor has recently been reported (Garrett et al., Nature, 394: 395-399 (1998)).
Unlike insulin, the activity and half-life of IGF-I are modulated by six IGF-I binding proteins (IGFBP""s 1-6), and perhaps additionally by a more distantly-related class of proteins (Jones and Clemmons, supra; Baxter et al., Endocrinology, 139: 4036 (1998)). IGFBP""s can either inhibit or potentiate IGF activity, depending on whether they are soluble or cell-membrane associated (Bach and Rechler, Diabetes Reviews, 3: 38-61 (1995)). The IGFBPs bind IGF-I and IGF-II with varying affinities and specificities (Jones and Clemmons, supra; Bach and Rechler, supra). For example, IGFBP-3 binds IGF-I and IGF-II with a similar affinity, whereas IGFBP-2 and IGFBP-6 bind IGF-II with a much higher affinity than they bind IGF-I (Bach and Rechler, supra; Oh et al., Endocrinology, 132, 1337-1344 (1993)).
The classical IGFBP""s have a molecular mass ranging from 22-31 kDa and contain a total of 16-20 cysteines in their conserved amino- and carboxy-terminal domains (Bach and Rechler, supra; Clemmons, Cytokine Growth Factor Rev., 8: 45-62 (1997); Martin and Baxter, Curr. Op. Endocrinol. Diab., 16-21 (1994)). The central domain connecting both cysteine-rich regions is only weakly conserved and contains the cleavage sites for IGFBP-specific proteases (Chemausek et al, J. Biol. Chem., 270: 11377-11382 (1995); Clemmons, supra; Conover, Prog. Growth Factor Res., 6: 301-309 (1995)). Further regulation of the IGFBP""s may be achieved by phosphorylation and glycosylation (Bach and Rechler supra; Clemmons, supra). There is no high-resolution structure available for any intact member of the IGFBP family. However, the NMR structures of two N-terminal fragments from IGFBP-5 that retain IGF-binding activity have recently been reported (Kalus et al., EMBO J., 17: 6558-6572 (1998)).
IGF-I is a single-chain 70-amino-acid protein with high homology to proinsulin. Unlike the other members of the insulin superfamily, the C region of the IGF""s is not proteolytically removed after translation. The solution NMR structures of IGF-I (Cooke et al., Biochemistry 30: 5484-5491 (1991); Hua et al., J. Mol. Biol., 259: 297-313 (1996)), mini-IGF-I (an engineered variant lacking the C-chain; DeWolf et al., Protein Science, 5: 2193-2202(1996)), and IGF-II (Terasawa et al, EMBO J., 13: 5590-5597(1994); Torres et al., J. Mol. Biol., 248 385-401 (1995)) have been reported. It is generally accepted that distinct epitopes on IGF-I are used to bind receptor and binding proteins. It has been demonstrated in animal models that receptor-inactive IGF mutants are able to displace endogenous IGF-I from binding proteins and hereby generate a net IGF-I effect in vivo (Loddick et al., Proc. Natl. Acad. Sci. USA, 95: 1894-1898 (1998); Lowman et al., Biochemistry, 37: 8870-8878 (1998)) While residues Y24, Y29, Y31, and Y60 are implicated in receptor binding, IGF mutants thereof still bind to IGFBPs (Bayne et al, J. Biol. Chem., 265: 15648-15652 (1990); Bayne et al, J. Biol. Chem., 264: 11004-11008 (1989); Cascieri et al, Biochemistry, 27: 3229-3233 (1988); Lowman et al., supra.
Additionally, a variant designated (1-27,gly4,38-70)-hIGF-I, wherein residues 28-37 of the C region human IGF-I are replaced by a four-residue glycine bridge, has been discovered that binds to IGFBP""s but not to IGF receptors (Bar et al, Endocrinology 127: 3243-3245 (1990)).
A multitude of mutagenesis studies have addressed the characterization of the IGFBP-binding epitope on IGF-I (Bagley et al., Biochem.J., 259: 665-671 (1989); Baxter et al., J. Biol. Chem.,267: 60-65(1992); Bayne et al., J. Biol. Chem., 263: 6233-6239 (1988); Clemmons et al., J. Biol. Chem., 265: 12210-12216 (1990); Clemmons et al., Endocrinology, 131: 890-895 (1992); Oh et al., supra). In summary, the N-terminal residues 3 and 4 and the helical region comprising residues 8-17 were found to be important for binding to the IGFBP""s. Additionally, an epitope involving residues 49-51 in binding to IGFBP-1, -2 and -5 has been identified (Clemmons et al., Endocrinology, supra, 1992). Furthermore, a naturally occurring truncated form of IGF-I lacking the first three N-terminal amino acids (called des(1-3)-IGF-I) was demonstrated to bind IGFBP-3 with 25 times lower affinity(Heding et al, J. Biol. Chem., 271: 13948-13952 (1996); U.S. Pat. Nos. 5,077,276; 5,164,370; 5,470,828).
In an attempt to characterize the binding contributions of exposed amino acid residues in the N-terminal helix, several alanine mutants of IGF-I were constructed (Jansson et al., Biochemistry, 36: 4108-4117 (1997)). However, the circular dichroism spectra of these mutant proteins showed structural changes compared to wild-type IGF-I, making it difficult to clearly assign IGFBP-binding contributions to the mutated side chains. A different approach was taken in a very recent study where the IGFBP-1 binding epitope on IGF-I was probed by heteronuclear NMR spectroscopy (Jansson et al., J. Biol. Chem., 273: 24701-24707 (1998)). The authors additionally identified residues R36, R37 and R50 to be functionally involved in binding to IGFBP-1.
Other IGF-I variants have been disclosed. For example, in the patent literature, WO 96/33216 describes a truncated variant having residues 1-69 of authentic IGF-I. EP 742,228 discloses two-chain IGF-I superagonists which are derivatives of the naturally occurring single-chain IGF-I having an abbreviated C domain. The IGF-I analogs are of the formula: BCn,A wherein B is the B domain of IGF-I or a functional analog thereof, C is the C domain of IGF-I or a functional analog thereof, n is the number of amino acids in the C domain and is from about 6 to about 12, and A is the A domain of IGF-I or a functional analog thereof.
Additionally, Cascieri et al., Biochemistry, 27: 3229-3233 (1988) discloses four mutants of IGF-I, three of which have reduced affinity to the Type 1 IGF receptor. These mutants are: (Phe23, Phe24, Tyr25)IGF-I (which is equipotent to human IGF-I in its affinity to the Types 1 and 2 IGF and insulin receptors), (Leu24)IGF-I and (Ser24)IGF-I (which have a lower affinity than IGF-I to the human placental Type 1 IGF receptor, the placental insulin receptor, and the Type 1 IGF receptor of rat and mouse cells), and desoctapeptide (Leu24)IGF-I (in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF-I, which has lower affinity than (Leu24)IGF-I for the Type 1 receptor and higher affinity for the insulin receptor). These four mutants have normal affinities for human serum binding proteins.
Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988) discloses three structural analogs of IGF-I: (1-62)IGF-I, which lacks the carboxyl-terminal 8-amino-acid D region of IGF-I; (1-27,Gly4,38-70)IGF-I, in which residues 28-37 of the C region of IGF-I are replaced by a four-residue glycine bridge; and (1-27,Gly4,38-62) IGF-I, with a C region glycine replacement and a D region deletion. Peterkofsky et al., Endocrinology, 128: 1769-1779 (1991) discloses data using the Gly4 mutant of Bayne et al., supra, Vol. 264. U.S. Pat. No. 5,714,460 refers to using IGF-I or a compound that increases the active concentration of IGF-I to treat neural damage.
Cascieri et al., J. Biol. Chem., 264: 2199-2202 (1989) discloses three IGF-I analogs in which specific residues in the A region of IGF-I are replaced with the corresponding residues in the A chain of insulin. The analogs are: (Ile41,Glu45,Gln46,Thr49,Ser50,Ile51,Ser53,Tyr,55,Gln56)IGF-I, an A chain mutant in which residue 41 is changed from threonine to isoleucine and residues 42-56 of the A region are replaced; (Thr49,Ser50,Ile51)IGF-I; and (Tyr55, Gln56)IGF-I.
WO 94/04569 discloses a specific binding molecule, other than a natural IGFBP, that is capable of binding to IGF-I and can enhance the biological activity of IGF-I. WO98/45427 published Oct. 15,1998 and Lowman et al., supra, disclose IGF-I agonists identified by phage display. Also, WO 97/39032 discloses ligand inhibitors of IGFBP""s and methods for their use.
There are various forms of human insulin on the market that differ in the duration of action and onset of action, but have the native human sequence. Jens Brange, Galenics of Insulin, The Physico-chemical and Pharmaceutical Aspects of Insulin and Insulin Preparations (Springer-Verlag, New York, 1987), page 17-40. Regular insulin is a clear neutral solution that contains hexameric insulin. It is short acting, its onset of action occurs in 0.5 hour after injection and duration of action is about 6-8 hours. NPH (Neutral Protamine Hagedorn) insulin, also called Isophane Insulin, is a crystal suspension of insulin-protamine complex. These crystals contain approximately 0.9 molecules of protamine and two zinc atoms per insulin hexamer. Dodd et al., Pharmaceutical Research, 12: 60-68 (1995). NPH-insulin is an intermediate-acting insulin; its onset of action occurs in 1.5 hours and its duration of action is 18-26 hours. 70/30 insulin is composed of 70% NPH-insulin and 30% Regular insulin. There are also Semilente insulin (amorphous precipitate of zinc insulin complex), UltraLente insulin (zinc insulin crystal suspension), and Lente insulin (a 3:7 mixture of amorphous and crystalline insulin particles). Of the various types of insulins available, NPH-, 70/30, and Regular insulin are the most widely used insulins, accounting for 36%, 28%, and 15%, respectively, of the insulin prescriptions in 1996.
The use of recombinant DNA technology and peptide chemistry have allowed the generation of insulin analogs with a wide variety of amino acid substitutions, and IGF-like modifications to insulin have been made for the purpose of modifying insulin pharmacokinetics (Brange et al., Nature, 333: 679 (1988); Kang et al., Diabetes Care, 14: 571 (1991); DiMarchi et al., xe2x80x9cSynthesis of a fast-acting insulin analog based upon structural homology with insulin-like growth factor-I,xe2x80x9d in: Peptides: Chemistry and Biology, Proceedings of the Twelfth American Peptide Symposium, J. A. Smith and J. E. Rivier, eds. (ESCOM, Leiden, 1992), pp. 26-28; Weiss et al., Biochemistry,30: 7373 (1991); Howey et al., Diabetes, 40: (Supp 1) 423A (1991); Slieker and Sundell, Diabetes, 40: (Supp 1) 168A (1991); Cara et al., J. Biol. Chem.,265: 17820 (1990); Wolpert et al., Diabetes, 39: (Supp 1) 140A (1990); Bornfeldt et al., Diabetologia, 34: 307 (1991); Drejer, Diabetes/Metabolism Reviews, 8: 259 (1992); Slieker et al., Adv. Experimental Med. Biol., 343: 25-32 (1994)). One example of such an insulin analog is Humalog(trademark) insulin (rapid-acting monomeric insulin solution, as a result of reversing the Lys (B28) and Pro(B29) amino acids on the insulin B-chain) that was recently introduced into the market by Eli Lilly and Company. A review of the recent insulin mutants in clinical trials and on the market is found in Barnett and Owens, Lancet, 349: 47-51 (1997).
Slieker et al., 1994, supra, describe the binding affinity of various IGF and insulin variants to IGFBPs, IGF receptor, and insulin receptor, and in particular sought to confer IGFBP-binding ability to insulin through several combinations of mutations, including: (Phe38, Arg39, Ser40) insulin, (Glu4, Gln16, Phe17) insulin, and (Glu4, Gln16, Phe17, Phe38, Arg39, Ser40) insulin (the numbering of mature insulin used herein consists of consecutive numbering in the B chain (residues 1-30), followed by consecutive numbering in the A chain (residues 31-51); these correspond to residues numbered 1-30 and residues 66-86, respectively of proinsulin; cf. FIG. 4 herein). However, only weak affinity was found for these variants binding to the IGF binding proteins and insulin-receptor affinity was reduced as compared with wild-type insulin (Slieker et al., supra).
Although earlier reports could not find any affinity of insulin for the binding proteins, a group has measured a weak affinity of 251+/xe2x88x9291 nM of insulin for IGFBP-3 by BIAcore(trademark) experiments (Heding et al., supra).
Despite all these efforts, the view of the IGFBP-binding epitope on IGF-I has remained diffuse and at low resolution. The previous studies most often involved insertions of homologous insulin regions into IGF-I or protein truncations (e. g. des(1-3)-IGF-I), not differentiating between effects attributed to misfolding and real binding determinants. Combining the results of all these studies is further complicated by the fact that different techniques were used to analyze complex formation of the mutant IGF forms with the IGFBP""s, ranging from radiolabeled ligand binding assays to biosensor analysis.
There is a need in the art for molecules that act as IGF or insulin agonists, and also for molecules that binds to IGF binding proteins with high affinity and specificity for therapeutic or diagnostic purposes.
Accordingly, in one embodiment, the invention provides an IGF-I variant wherein an amino acid at position 3, 4, 5, 7, 10, 14, 17, 23, 24, 25, 43, 49 or 63, or any of such amino acids in combination with an amino acid at position 12 or 16 or both 12 and 16 of native-sequence human IGF-I, or any combination thereof, is replaced with any amino acid at said position 7 or with an alanine, a glycine, or a serine residue at any position other than said position 7.
In one preferred embodiment, the amino acids at said positions 16 and 49 are replaced to obtain binders to IGFBP-3. Another preferred embodiment for obtaining binders to IGFBP-3 is a variant containing mutations at positions 3 and 7.
In a still further preferred embodiment, additionally tyrosine at said position 24 is replaced with leucine or tyrosine at said position 31 is replaced with alanine or both are replaced, to disrupt or prevent receptor binding. Most preferably, both tyrosines at said positions 24 and 31 are replaced.
In another embodiment, the invention provides a long-half-life IGF-like insulin wherein phenylalanine at position 1 of native-sequence human pro-insulin is deleted (des(1)-proinsulin), or glutamine at position 4 of native-sequence human pro-insulin is replaced with glutamic acid, or leucine at position 17 of native-sequence human pro-insulin is replaced with phenylalanine, or phenylalanine at position 25 of native-sequence human pro-insulin is replaced with tyrosine, or tyrosine at position 26 of native-sequence human pro-insulin is replaced with phenylalanine, or threonine at position 73 of native-sequence human pro-insulin is replaced with phenylalanine, or any combination thereof.
Preferably, for the IGF-like insulin, amino acids at said positions 4, 17, 26, and/or 73 are replaced to generate IGFBP-1-specific mutants, or the amino acid at position 1 is deleted and the amino acids at positions 25, 26, and/or 73 are replaced to generate IGFBP-3-specific mutants.
In yet another embodiment, the invention provides an IGF-like insulin wherein the phenylalanine at position 1 is deleted (des(1)-insulin), or glutamine at position 4 of native-sequence human mature insulin is replaced with glutamic acid, or leucine at position 17 of native-sequence human mature insulin is replaced with phenylalanine, or phenylalanine at position 25 of native-sequence human mature insulin is replaced with tyrosine, or tyrosine at position 26 of native-sequence human mature insulin is replaced with phenylalanine, or threonine at position 38 of native-sequence human mature insulin is replaced with phenylalanine, or any combination thereof (Note: the numbering of mature insulin used here consists of consecutive numbering in the B chain (residues 1-30), followed by consecutive numbering in the A chain (residues 31-51)).
In a preferred embodiment, amino acids of the above mature insulin at positions 4, 17, 26, and 38 are replaced, to create a mutant that is IGFBP-1 specific.
In another preferred embodiment, the amino acid at position 1 of the above mature insulin is deleted, and amino acids of the above mature insulin at positions 25, 26, and 38 are replaced, to create a mutant that is IGFBP-3 specific.
Also provided herein is a composition comprising one of the peptides described above in a carrier, preferably a pharmaceutically acceptable carrier. Preferably, this composition is sterile.
Uses of these peptides include all uses that liberate or enhance at least one biological activity of exogenous or endogenous IGFs or insulin. They can be used in treating, inhibiting, or preventing conditions in which an IGF such as IGF-I or insulin is useful, i.e., in treating an IGF disorder or an insulin disorder by administering an effective amount of the peptide to a mammal, as described below.
Additionally provided herein is a method for increasing serum and tissue levels of biologically active IGF or insulin in a mammal comprising administering to the mammal an effective amount of a peptide as described above. The mammal is preferably human. Also preferred is where administering the peptide, if it is mimicking IGF-I, preferably in an amount effective to produce body weight gain, causes an increase in anabolism in the mammal. Additionally preferred is that glycemic control is effected in the mammal after the peptide is administered.
The peptide herein can be administered alone or together with another agent such as GH, a GH-releasing peptide (GHRP), a GH-releasing factor (GHRF), a GH-releasing hormone (GHRH),a GH secretagogue, an IGF, an IGF in combination with an IGFBP, an IGFBP, GH in combination with a GH binding protein (GHBP), insulin, or a hypoglycemic agent (which includes in the definition below an insulin-sensitizing agent such as thiazolidinedione).
In yet another aspect of the invention, a method is provided for effecting glycemic control in a mammal comprising administering to the mammal an effective amount of one or more of the above peptides. Preferably, the peptide also reduces plasma insulin secretion and blood glucose levels in a mammal. Also preferably, the mammal has a hyperglycemic disorder such as diabetes. This method can additionally comprise administering to the mammal an effective amount of a hypoglycemic agent or insulin.
Also provided is a method for increasing serum and tissue levels of biologically active IGF in a mammal, or a method for increasing anabolism in a mammal, or a method for controlling glycemia in a mammal comprising administering to the mammal an effective amount of the composition containing the peptide herein.
Also contemplated herein is a kit comprising a container containing a pharmaceutical composition containing the peptide herein and instructions directing the user to utilize the composition. This kit may optionally further comprise a container containing a GH, a GHRP, a GHRF, a GHRH, a GH secretagogue, an IGF, an IGF complexed to an IGFBP, an IGFBP, a GH complexed with a GHBP, insulin, or a hypoglycemic agent.
For an identification of the peptides herein, human IGF-I was displayed monovalently on filamentous phagemid particles (U.S. Pat. Nos. 5,750,373 and 5,821,047), and a complete alanine-scanning mutagenesis thereof (Cunningham and Wells, Science, 244: 1081-1085 (1989); U.S. Pat. No. 5,834,250) was performed by phage display (xe2x80x9cturbo-ala scanxe2x80x9d) (Cunningham et al., EMBO J., 13: 2508-2515 (1994); Lowman, Methods Mol. Biol., 87: 249-264 (1998)). The mutant IGF-phagemids were used to map the binding determinants on IGF-I for IGFBP-1 and IGFBP-3. The alanine scanning reveals specificity determinants for these binding proteins, so as to generate binding-protein-specific IGF variants or insulin variants that bind specifically to IGFBP-1 or IGFBP-3 to modulate their clearance half-life, improve proteolytic stability, or alter their tissue distribution in vivo. These mutants should also be useful for mapping the functional binding site for IGF receptor, whose crystal structure was recently reported (Garrett et al., supra). In addition, it may be of interest to map the epitopes of various IGF-binding antibodies or of other peptides or proteins that bind to IGF-I.